This project will contribute information needed to describe and ultimately to understand one of the major stages in the expression of genetic information, namely, RNA splicing. We have generated antibodies for a novel branch structure (an RNA lariat) that is a key intermediate formed during intron removal from mRNA precursor molecules of eukaryotes. These antibodies will be used to develop assays that can detect and quantitate the RNA lariat intermediates that form during splicing in nuclear extracts. Antibodies will also be used to probe the lariat structures form from a variety of introns where differences in the branch consensus sequences exist. Anti-branch antibodies will be tested as specific inhibitors of splicing since they recognize the first product of the splicing reaction and would allow the separation of the two major steps of the splicing reaction. A second project will use RNA splicing to map introns in genomic DNA restriction fragments. The strategy depends on generating RNA from DNA fragments cloned into a transcription vector into which either intact 3' or 5' splice sites have been engineered. Two vectors will be designed, one supplying an intact 3' and another an intact 5' splice site. Each could complement an opposite splice site within the cloned genomic insert to produce a chimaeric RNA with a spliceable intron. An intact splice site inserted downstream from each of the independently activatable promoters in the transcription vector will reveal the strand of the cloned DNA that contains this splice site. An unrelated project will attempt to find specific proteins that bind to the oligo U sequences found in the 3' untranslated regions of many eukaryotic messenger RNAs (mRNA). The existence of such proteins would suggest that oligo U sequences share some common function in mRNAs. Such proteins would be identified initially by their binding to poly U dated to cytoplasmic extracts. If specific proteins are identified, their binding to the oligo U(+) mRNA population would be tested and ultimately binding to an oligo U containing mRNA such as beta-actin would be tested.